Method and apparatus for sorting particles

ABSTRACT

A method and apparatus for sorting particles moving through a closed channel system of capillary size comprises a bubble valve for selectively generating a pressure pulse to separate a particle having a predetermined characteristic from a stream of particles. The particle sorting system may further include a buffer for absorbing the pressure pulse. The particle sorting system may include a plurality of closely coupled sorting modules which are combined to further increase the sorting rate. The particle sorting system may comprise a multi-stage sorting device for serially sorting streams of particles, in order to decrease the error rate.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/499,254 filed Jul. 8, 2009 which, in turn, is a continuation of U.S.patent application Ser. No. 11/101,038, filed Apr. 6, 2005, which, inturn, is a divisional of 10/329,008, filed Dec. 23, 2002, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/411,058,filed Sep. 16, 2002, and is a continuation-in-part of U.S. patentapplication Ser. No. 10/179,488, filed Jun. 24, 2002, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/373,256,filed Apr. 17, 2002, the contents of each application is incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a method and apparatus for the sorting ofparticles in a suspension, where the input flow path of a sorting modulecan be split into several output channels. More particular, theinvention relates to a particle sorting system in which a plurality ofsorting modules are interconnected as to yield an increased particlethroughput.

BACKGROUND OF THE INVENTION

In the fields of biotechnology, and especially cytology and drugscreening, there is a need for high throughput sorting of particles.Examples of particles that require sorting are various types of cells,such as blood platelets, white blood cells, tumorous cells, embryoniccells and the like. These particles are especially of interest in thefield of cytology. Other particles are (macro) molecular species such asproteins, enzymes and poly-nucleotides. This family of particles is ofparticular interest in the field of drug screening during thedevelopment of new drugs.

Methods and apparatus for particle sorting are known, and the majoritydescribed in the prior art work in the condition where the particles aresuspended in a liquid flowing through a channel network having at leasta branch point downstream and are operated according to thedetect-decide-deflect principle. The moving particle is first analyzedfor a specific characteristic, such as optical absorption, fluorescentintensity, size etc. Depending on the outcome of this detection phase,it is decided how the particle will be handled further downstream. Theoutcome of the decision is then applied to deflect the direction ofspecific particle towards a predetermined branch of the channel network.

Of importance is the throughput of the sorting apparatus, i.e. how manyparticles can be sorted per unit of time. Typical sorting rates forsorters employing flows of particle suspension in closed channels are inthe range from a few hundred particles per second to thousands ofparticles per second, for a single sorting unit.

An example of a sorting device is described in U.S. Pat. No. 4,175,662,the contents of which are herein incorporated by reference (hereinafterreferred to as the '662 patent). In the '662 patent, a flow ofparticles, cells in this case, flows through the center of a straightchannel, which branches into two perpendicular channels at a branchingpoint downstream (T-branch). The entering particles are surrounded by asheath of compatible liquid, keeping the particles confined to thecenter of the channel. In normal conditions, the flow ratio through thetwo branches is adjusted so that the particles automatically flowthrough one of the branches. In a section of the channel acharacteristic of the particles is determined using a detector, whichcan be an optical system (detection phase). The detector generates asignal when the detector detects a particle possessing a predeterminedcharacteristic in the decision phase. Once a particle is detected, adeflector is activated for deflecting the particle in a deflectionphase. In this case, the deflector comprises an electrode pair,positioned in the branch of the channel where the particles normallyflow through in the inactivated state of the deflector. By theapplication of current pulses, the aqueous liquid is electrolysed,yielding a gas bubble evolving between the electrode pair. As the gasbubble increases in size, the flow rate through this branch is reducedduring the evolving phase. After the current pulse is applied, thebubble growth stops and the gas bubble is carried along with the flow.As a result, the flow through the specific branch is momentarily reducedand the particle of interest changes paths and flows down the otherbranch.

The device of the '662 patent is effective for sorting particles.However one serious drawback is that gas bubbles are created whichpotentially can accumulate at certain points of the fluidic network.This bubble generation can clog the flow channels, yielding erroneoussorting. Another drawback is that the generated gasses (mostly oxygenand hydrogen) and ionic species (mostly OH⁻ and H⁺) influence theparticles flowing through the branch with the electrode pair. Inaddition, cells and delicate proteins such as enzymes are very fragileand can be destroyed by the fouling constituents co-generated with thegas bubble. Another drawback is the complexity of the overall sortingapparatus. In particular, the micro electrode construction is verycomplex to mount and assemble in the small channels of the system. As aresult, the cost of a sorting unit is relatively large.

Another example of a particle sorting system of the prior art isdisclosed in U.S. Pat. No. 3,984,307, the contents of which are hereinincorporated by reference (hereinafter the '307 patent). In the '307patent, the particles are flowing, confined by a flowing sheath liquid,through the center of a channel. After passing a detector section, thechannel branches into two channels forming an acute angle therebetween(e.g., Y-branch). Just before the branching point, an electricallyactivated transducer is located in the channel for deflecting a specificparticle having an appropriate, predetermined characteristic. Thetransducer described is a piezo actuator or ultrasonic transducer,yielding upon electrical activation a pressure wave in the channel. Thegenerated pressure wave momentarily disturbs the flow in one branch thusdeflecting the particle of interest into the other branch.

In the device of the '307 patent, as in the previous discussed device,the deflector is incorporated within the channel system, resulting inrelatively large construction costs. Another drawback of this device isthe deflector principle used. The generated pressure waves are notconfined to the branching point, but rather propagate upstream into thedetector section, as well as down both branches. This influences theoverall flow through the channel. This is particularly a drawback ifsorters of this type are connected either in series or in parallel, asis typically done to construct a high throughput sorting system.Pressure waves generated in one sorter can then influence the flows anddeflection of particles in neighboring sorter units.

Another sorter is described in U.S. Pat. No. 4,756,427, the contents ofwhich are herein incorporated by reference. This sorter is analogous tothe sorter in the '662 patent. In this case, however, the flow in onebranch is disturbed by momentarily changing the resistance of thebranch. The resistance is changed by changing the height of the branchchannel by an external actuator. In the preferred embodiment, thisexternal actuator is a piezo disc glued on top of the channel, causingit to move downwards upon activation.

Although the construction of the sorter described in the '427 patent isless complex than the previously described sorter structures, it isstill problematic to couple multiple sorter modules of the describedtype together to increase the sorting rate. This is, as in the sorterdescribed in the '307 patent because of the generated pressure wavescausing interference with other sorter modules.

Another particle sorting device is described in U.S. Pat. No. 5,837,200,the contents of which are herein incorporated by reference. The '200patent describes a sorting device that uses a magnetic deflection moduleto classify or select particles based on their magnetic properties. The'200 patent further describes processing and separating individualparticle streams in parallel.

SUMMARY OF THE INVENTION

The present invention provides a method and apparatus for sortingparticles moving through a closed channel system of capillary size. Theparticle sorting system of the invention provides a sorting module thatcan be assembled at low cost while providing an accurate means ofsorting large amounts of particles per unit of time. The particlesorting system may include a plurality of closely coupled sortingmodules which are combined to further increase the sorting rate. Theparticle sorting system may comprise a multi-stage sorting device forserially sorting streams of particles, in order to decrease the errorrate.

The particle sorting system implements an improved fluidic particleswitching method and switching device according to the presentinvention. The particle sorting system comprises a closed channel systemof capillary size for sorting particles. The channel system comprises afirst supply duct for introducing a stream of particles and a secondsupply duct for supplying a carrier liquid. The first supply duct formsa nozzle to introduce a stream of particles into the flow of carrierliquid. The first supply duct and the second supply duct are in fluidcommunication with a measurement duct, which branches into a firstbranch and a second branch at a branch point. A measurement region isdefined in the measurement duct and is associated with a detector tosense a predetermined characteristic of particles in the measurementregion. Two opposed bubble valves are positioned in communication withthe measurement duct and are spaced opposite each other. The bubblevalves communicate with the measurement duct through a pair of opposedside passages. Liquid is allowed to partly fill these side passages toform a meniscus therein which interfaces the carrier liquid with thereservoir of the bubble valves. An external actuator is also providedfor actuating one of the bubble valves. When the external actuator isactivated, the pressure in the reservoir of the activated bubble valveincreases, deflecting the meniscus and causing a flow disturbance in themeasurement duct to deflect the flow therein.

When a sensor located in the measuring region senses a predeterminedcharacteristic in a particle flowing through the measurement region, thesensor produces a signal in response to the sensed characteristic. Theexternal actuator is responsive to the sensor to cause a pressure pulsein a compression chamber of a first bubble valve to deflect the particlewith the predetermined characteristic, causing the selected particle toflow down the second branch duct.

In one aspect, the invention comprises a method of sorting particlesincluding the steps of providing a measurement duct having an inlet anda branching point at which the duct separates into two branch ducts, andconducting a stream of fluid into the duct inlet with a stream ofparticles suspended therein, such that the particles normally flowthrough a first one of the branch ducts and providing upstream from thebranching point two opposing side passages for momentarily deflectingthe stream in the duct. A first one of the side passages ishydraulically connected to a compression chamber of a first bubblevalve, which is acted upon by an external actuator for varying thepressure therein. A second of the side passages is hydraulicallyconnected with a buffer chamber of a second bubble valve for absorbingpressure variations. The method further comprises providing ameasurement station along the measurement duct upstream of the sidepassages for sensing a predetermined characteristic of particles in thestream and for producing a signal when the predetermined characteristicis sensed. The method further comprises the step of, in response tosensing the predetermined characteristic, activating the externalactuator for creating a flow disturbance in the duct between the sidepassages, thereby deflecting the particle having the predeterminedcharacteristics and causing the selected particle to flow down thesecond branch duct.

In further aspects of the invention, the particle sort rate isrespectively increased or the type of particles sorted being increased,by respectively connecting a plurality of sorting modules in parallel orserially connecting a plurality of sorting modules in a binary tree likeconfiguration.

According to one aspect of the invention, a particle sorting system isprovided. The particles sorting system comprises a first duct forconveying a stream of suspended particles confined in a carrier liquid,comprising an inlet, a first outlet and a second outlet, a sensor forsensing a predetermined characteristic in a particle, a side channel incommunication with the first duct, a sealed chamber positioned adjacentto the side channel, wherein the carrier fluid forms a meniscus in theside channel to separate the sealed chamber from the carrier fluid; andan actuator. The actuator modifies the pressure in the sealed chamber todeflect the meniscus when the sensor senses the predeterminedcharacteristic. The deflection of the meniscus causes the particlehaving the predetermined characteristic to flow into the second outletwhile particles that do not have the predetermined characteristic flowinto the first outlet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a particle sorting system according to anillustrative embodiment of the invention.

FIGS. 2 through 4 illustrate the operation of the particle sortingsystem of FIG. 1.

FIG. 5 illustrates a particle sorting system showing alternate positionsfor the actuator chamber and the buffer chamber.

FIG. 6 illustrates the particle sorting system according to anotherembodiment of the invention.

FIG. 7 illustrates a bubble valve suitable for use in the particlesorting system of the present invention.

FIG. 8 is a schematic diagram of the particle sorting system of anillustrative embodiment of the present invention.

FIG. 9 shows one embodiment of a particle sorting system for sortingparallel streams of particles according to the teachings of the presentinvention.

FIG. 10 shows one embodiment of a particle sorting system configured ina binary tree-like configuration of sorting modules according to theteachings of the present invention.

FIG. 11 illustrates another embodiment of a multi-stage particle sortingsystem for sorting parallel streams of particles in multiple stages.

FIG. 12 illustrates a parallel particle sorting system according to analternate embodiment of the present invention.

FIG. 13 illustrates a parallel particle sorting system according toanother embodiment of the present invention.

FIGS. 14 a and 14 b illustrate a particle sorting system according toanother embodiment of the invention, including an optical mask to allowmeasurement of a particle size and/or velocity.

FIG. 15 illustrates a parallel sorting system having variable channelsaccording to another embodiment of the present invention.

FIG. 16 illustrates a variable array design of a parallel sorting systemaccording to another embodiment of the present invention.

FIG. 17 illustrates a parallel sorting system according to anotherembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a particle sorting system for sortingparticles suspended in a liquid. The particle sorting system provideshigh-throughput, low error sorting of particles based on a predeterminedcharacteristic. The present invention will be described below relativeto illustrative embodiments. Those skilled in the art will appreciatethat the present invention may be implemented in a number of differentapplications and embodiments and is not specifically limited in itsapplication to the particular embodiments depicted herein.

The terms “duct” “channel” and “flow channel” as used herein refers to apathway formed in or through a medium that allows for movement offluids, such as liquids and gases. The channel in the microfluidicsystem preferably have cross-sectional dimensions in the range betweenabout 1.0 μm and about 500 μm, preferably between about 25 μm and about250 μm and most preferably between about 50 μm and about 150 μm. One ofordinary skill in the art will be able to determine an appropriatevolume and length of the flow channel. The ranges are intended toinclude the above-recited values as upper or lower limits The flowchannel can have any selected shape or arrangement, examples of whichinclude a linear or non-linear configuration and a U-shapedconfiguration.

The term “particle” refers to a discrete unit of matter, including, butnot limited to cells.

The term “sensor” as used herein refers to a device for measuring acharacteristic of an object, such as a particle.

The term “bubble valve” as used herein refers to a device that generatespressure pulses to control flow through a channel.

The term “carrier fluid” as used herein refers to a sheath of compatibleliquid surrounding a particle for carrying one or more particles througha duct or channel.

FIG. 1 is a schematic depiction of a particle sorting system 10according to the teachings of the present invention. According to oneapplication of the present invention, the particle sorting system 10comprises a closed channel system of capillary size for sortingparticles. The channel system comprises a first supply duct 12 forintroducing a stream of particles 18 and a second supply duct 14 forsupplying a carrier liquid. The first supply duct 12 forms a nozzle 12a, and a stream of particles is introduced into the flow of the carrierliquid. The first supply duct 12 and the second supply duct 14 are influid communication with a measurement duct 16 for conveying theparticles suspended in the carrier liquid. The measurement duct branchesinto a first branch channel 22 a and a second branch channel 22 b at abranch point 21. A measurement region 20 is defined in the measurementduct 16 and is associated with a detector 19 to sense a predeterminedcharacteristic of the particles passing through the measurement region20. Two opposed bubble valves 100 a and 100 b are positioned relative tothe measurement duct and disposed in fluid communication therewith. Thevalves are spaced opposite each other, although those of ordinary skillwill realize that other configurations can also be used. The bubblevalves 100 a and 100 b communicate with the measurement duct 16 througha pair of opposed side passages 24 a and 24 b, respectively. Liquid isallowed to partly fill these side passages 24 a and 24 b to form ameniscus 25 therein. The meniscus defines an interface between thecarrier liquid and another fluid, such as a gas in the reservoir of theassociated bubble valve 100. An actuator 26 is also provided foractuating either bubble valve, which momentarily causes a flowdisturbance in the duct to deflect the flow therein when activated bythe actuator 26. As illustrated, the actuator is coupled to the bubblevalve 100 b. The second bubble valve 100 a serves as a buffer forabsorbing the pressure pulse created by the first bubble valve 100 b.

The first side passage 24 b is hydraulically connected to a compressionchamber 70 b in the first bubble valve 100 b, so that if the pressure inthis chamber is increased, the flow in the measurement duct near theside passage is displaced inwards, substantially perpendicular to thenormal flow in the duct. The second side passage 24 a, positionedopposite of the first side passage 24 b is hydraulically connected to abuffer chamber 70 a in the second bubble valve 100 a for absorbingpressure transients. This first side passage 24 b co-operates with thesecond side passage 24 a to direct the before mentioned liquiddisplacement caused by pressurizing the compression chamber 70 b, sothat the displacement has a component perpendicular to the normal flowof the particles through the measurement duct.

Upon pressurizing the compression chamber 70 b an amount of liquid istransiently discharged from the first side passage 24 b. The resiliencyof the second side passage 24 a results upon a pressurized discharge, ina transient flow of the liquid in the duct into the second side passage24 a. The co-operation of the two side passages and the fluidicstructures they interconnect causes the flow through the measurementduct 16 to be transiently moved sideways back and forth uponpressurizing and depressurising of the compression chamber 70 b inducedby the external actuator 26 in response to the signal raised by thedetection means 19. This transient liquid displacement, having acomponent perpendicular to the normal flow in the duct, can be appliedin deflecting particles having predetermined characteristics to separatethem from the remaining particles in the mixture.

As shown, the measurement duct 16 branches at the branch point 21 intotwo branches 22 a, 22 b and the flow rates in these branches areadjusted so that the particles normally stream through the second of thetwo branches 22 b. The angle between the branches 22 a, 22 b is between0 and 180 degrees, and preferably between 10 and 45 degrees. However,the angle can even be 0 degrees, which corresponds to two parallel ductswith a straight separation wall between them.

The particles to be sorted are preferably supplied to a measurementposition in a central fluid current, which is surrounded by a particlefree liquid sheath. The process of confining a particle stream is known,and often referred to as a ‘sheath flow’ configuration. Normally,confinement is achieved by injecting a stream of suspended particlesthrough a narrow outlet nozzle into a particle free carrier liquidflowing in the duct 16. By adjusting the ratio of flow rates of thesuspension and carrier liquid, the radial confinement in the duct aswell as the inter particle distance can be adjusted. A relatively largeflow rate of the carrier liquid results in a more confined particlestream having a large distance between the particles.

In a suspension introduced by the first supply duct 12, two types ofparticles can be distinguished, normal particles 18 a and particles ofinterest 18 b. Upon sensing the predetermined characteristic in aparticle 18 b in the measurement region 20, the detector 19 raises asignal. The external actuator 26 activates the first actuator bubblevalve 100 b, when signaled by the detector 19 in response to sensing thepredetermined characteristic, to create a flow disturbance in themeasurement duct 16 between the side passages 24 a, 24 b. The flowdisturbance deflects the particle 18 b having the predeterminedcharacteristic so that it flows down the first branch duct 22 a ratherthan the second branch duct 22 b. The detector communicates with theactuator 26, so that when the detector 19 senses a predeterminedcharacteristic in a particle, the actuator activates the first bubblevalve 100 b to cause pressure variations in the reservoir 70 b of thefirst bubble valve. The activation of the first bubble valves deflectsthe meniscus 25 b in the first bubble valve 100 b and causes a transientpressure variation in the first side passage 24 b. The second sidepassage 24 a and the second bubble valve 100 a absorb the transientpressure variations in the measurement duct 16 induced via the actuator26. Basically, the reservoir 70 a of the second bubble valve 100 a is abuffer chamber having a resilient wall or containing a compressiblefluid, such as a gas. The resilient properties allow the flow of liquidfrom the measurement duct into the second side passage 24 a, allowingthe pressure pulse to be absorbed and preventing disturbance to the flowof the non-selected particles in the stream of particles.

At the measurement region 20, individual particles are inspected, usinga suitable sensor 19, for a particular characteristic, such as size,form, fluorescent intensity, as well as other characteristics obvious toone of ordinary skill. Examples of applicable sensor, known in the art,are various types of optical detection systems such as microscopes,machine vision systems and electronic means for measuring electronicproperties of the particles. Particularly well known systems in thefield are systems for measuring the fluorescent intensity of particles.These systems comprise a light source having a suitable wavelength forinducing fluorescence and a detection system for measuring the intensityof the induced fluorescent light. This approach is often used incombination with particles that are labelled with a fluorescent marker,i.e. an attached molecule that upon illuminating with light of aparticular first wavelength produces light at another particular secondwavelength (fluorescence). If this second wavelength light is detected,the characteristic is sensed and a signal is raised.

Other examples include the measurement of light scattered by particlesflowing through the measurement region. Interpreting the scatteringyield information on the size and form of particles, which can beadopted to raise a signal when a predetermined characteristic isdetected.

The actuator 26 for pressurizing the compression chamber of the firstbubble valve can comprise an external actuator that responds to a signalfrom the sensor that a particle has a selected predeterminedcharacteristic. There are two classes of external actuators that aresuitable for increasing the pressure. The first class directly providesa gas pressure to the liquid in the first side passage 24 b. Forexample, the actuator may comprise a source of pressurized gas connectedwith a switching valve to the liquid column in the side passage 24 b.Activation of the switch connects the passage to the source ofpressurized gas, which deflects the meniscus in the liquid. Upondeactivation, the switch connects the passage 24 b back to the normaloperating pressure.

Alternatively, a displacement actuator may be used in combination with aclosed compression chamber having a movable wall. When the displacementactuator displaces the wall of the compression chamber inward, thepressure inside increases. If the movable wall is displaced back to theoriginal position, the pressure is reduced back to the normal operatingpressure. An example of a suitable displacement actuator is anelectromagnetic actuator, which causes displacement of a plunger uponenergizing a coil. Another example is the use of piezoelectric material,for example in the form of a cylinder or a stack of disks, which uponthe application of a voltage produces a linear displacement. Both typesof actuators engage the movable wall of the compression chamber 70 tocause pressure variations therein.

FIGS. 2 through 4 illustrate the switching operation of switch 40 in theparticle sorting system 10 of FIG. 1. In FIG. 2, the detector 19 sensesthe predetermined characteristic in a particle and generates a signal toactivate the actuator 26. Upon activation of the actuator, the pressurewithin the reservoir 70 b of the first bubble valve 100 b is increased,deflecting the meniscus 25 b and causing a transient discharge of liquidfrom the first side passage 24 b, as indicated by the arrow. The suddenpressure increase caused at this point in the duct causes liquid to flowinto the second side passage 24 a, because of the resilient propertiesof the reservoir of the second bubble valve 100 a. This movement ofliquid into the second side passage 24 a is indicated with an arrow. Asa result, as can be seen in the figure, the flow through the measurementduct 16 is deflected, causing the selected particle of interest 18 blocated between the first side passage 24 b and the second side passage24 a to be shifted perpendicular to its flow direction in the normalstate. The flow resistances to the measurement duct 16, the first branch22 a and the second branch 22 b is chosen so that the preferreddirection of the flow to and from the first side passage 24 b and thesecond side passage 24 a has an appreciable component perpendicular tothe normal flow through the measurement duct 16. This goal can forinstance be reached by the first branch 22 a and the second branch 22 bso that their resistances to flow is large in comparison with the flowresistances of the first side passage 24 b and the second side passage24 a.

FIG. 3 shows the particle sorting system 10 during the relief of thefirst bubble valve reservoir when the particle of interest 18 b has leftthe volume between the first side passage 24 b and the second sidepassage 24 a. The actuator 26 is deactivated, causing the pressureinside the reservoirs 70 a, 70 b to return to the normal pressure.During this relief phase there is a negative pressure difference betweenthe two reservoirs 70 a, 70 b of the bubble valves, causing a liquidflow through the first side passage 24 b and the second side passage 24a opposite to the liquid flow shown in the previous figure and asindicated by the arrows.

FIG. 4 illustrates the particle sorting system 10 after completion ofthe switching sequence. The pressures inside the reservoirs of thebubble valves are equalized, allowing the flow through the measurementduct 16 to normalize. As the particle of interest 18 b has beendisplaced radially, it will flow into the first branch 22 a, while theother particle continue to flow into the second branch 22 b, therebyseparating the particles based on the predetermined characteristic.

This process of detecting and selective deflecting of particles may berepeated many times per second for sorting particles at a high rate.Adopting the fluid switching as described, switching operations may beexecuted up to around several thousand switching operations per second,yielding sorting rates in the order of million sorted particles perhour.

According to another embodiment of the invention, the actuator bubblevalve 100 b and the buffer bubble valve 100 a may be placed in differentpositions. For example, as shown in FIG. 5, the actuator bubble valve100 b and the first side passage 24 b and/or the buffer bubble valve 100a and the second side passage 24 a may be place upstream from the branchpoint 21. The components may be placed in any suitable location, suchthat the flow resistance between the actuator chamber 70 b and thebuffer chamber 70 a is less than the flow resistance between any ofthese latter components and other pressure sources. More particularly,the actuator chamber 70 b and the buffer chamber 70 a may be placed suchthat the flow resistance between them is less than the flow resistancebetween a selected particle and a subsequent particle in the stream ofparticles. The positioning of the components in this manner thusprevents a pressure wave generated by the above-described method ofdeflecting a single selected particle, from travelling upstream ordownstream and affecting the flow of the remaining particles in thestream of particles. A larger difference in flow resistances results ina higher level of isolation of the fluidic switching operation withassociated pressure transients from the flow characteristics in the restof the system. Moreover, the in-situ dampening of generated pressurepulses applied for sorting allows the implementation of sorting networkscomprising a plurality of switches 40, each of which is hydraulicallyand pneumatically isolated from the others.

According to another embodiment, shown in FIG. 6, the particle sortingsystem of the present invention may use any suitable pressure wavegenerator (in place of a bubble valve) in combination one or more bubblevalves serving as a buffer, such as valve 100 b. For example, thepressure wave generator 260 may comprise an actuator such as apiezoelectric column or a stepper motor, provided with a plunger thatcan act upon the flowing liquid, either directly or via deflection ofthe channel system, to selectively deflect particles when the actuatoris activated by a signal. Other suitable pressure wave generatorsinclude electromagnetic actuators, thermopneumatic actuators and a heatpulse generator for generating vapor bubbles in the flowing liquid byapplying heat pulses. The buffer bubble valve 100 b is positioned toabsorb the pressure wave created by the pressure wave generator 260 toprevent flow disturbance in the other particles of the particle stream.The spring constant of the buffer 100 b may be varied according to theparticular requirements by varying the volume of the buffer chamber 70b, the cross-sectional area of the side passage 24 b and/or thestiffness or the thickness of a flexible membrane (reference 72 in FIG.7) forming the buffer chamber 70 b.

FIG. 7 illustrates an embodiment of a valve 100 suitable for creating apressure pulse to separate particles of interest from other particles ina stream of particles and/or acting as a buffer for absorbing a pressurepulse according to the teachings of the present invention. As shown, thevalve 100 is formed adjacent to a side passage 24 a or 24 b formed in asubstrate which leads to the measurement duct 16. The side passage 24 aincludes a fluid interface port 17 formed by an aperture in the sidewall of the passage. A sealed compression chamber 70 is positionedadjacent to the side passage 24 a and communicates with the side passagethrough the fluid interface port. The illustrative chamber 70 is formedby a seal 71 and a flexible membrane 72. The carrier fluid in the sidepassage 24 a forms a meniscus 25 at the interface between the sidepassage and the chamber. The actuator 26 depresses the flexible membraneto increase the pressure in the chamber, which deflects the meniscus andcauses a pressure pulse in the carrier fluid.

FIG. 8 shows a sorting module 50 having an appropriate supply duct 52for providing a stream of particles to be sorted as well as a firstoutlet duct 54 and a second outlet duct 56, either of which can carrythe particles sorted in the sorting module 50. The sorting module 50comprises a detector system 19 for sensing particles entering thesorting module 50 via the supply duct 52 can be operationally connectedto a switch 40 for providing the required switching capabilities to sortparticles. The first branch 22 b and the second branch 22 a, FIG. 1, canbe disposed in fluidic connection with the outlet duct 54 and the secondoutlet duct 56.

FIG. 9 shows a particle sorting system 500 according to an alternateembodiment of the invention, comprising a plurality of sorting modules50 that can be coupled together in any appropriate configuration. Forexample, the modules 50 in this embodiment are coupled in parallel. Theoutlet ducts 54 of the sorting modules 50 are coupled to a firstcombined outlet 58, the second outlet ducts 56 are coupled to a secondcombined outlet 60. The parallel arrangement of sorting modules yields asystem of combined sorting module 50 having an overall sorting rate of Ntimes the sorting rate of an individual sorting module 50, where N isthe number of parallel connected sorting module 50.

FIG. 10 shows a particle sorting system 550 according to anotherembodiment, comprising a first sorting module 50 a in series with asecond sorting module 50 b. The second sorting module 50 b may beequipped for sorting particles having a predetermined characteristic thesame or different than the predetermined characteristic of the particlessorted by the first sorting module 50 a. The particle stream enters thefirst sorting module 50 a through the supply duct 52 and may contain atleast two types of particles. A first type of particle is sorted in thefirst sorting module 50 a and exits through the first outlet duct 54 a.The remaining particles exit the first sorting module 50 a throughsecond outlet duct 56 a and are introduced into the second sortingmodule 50 b via the second supply duct 52 b. From this stream ofparticles, particles having the other predetermined characteristic aresorted and exit through the second outlet duct 54 b . Particles thatposses neither of the two predetermined characteristics exit the secondsorting module 50 b via the second outlet duct 56 b. Those of ordinaryskill will readily recognize that any suitable type of sorting module 50can be used, and can be coupled together in a variety of ways, dependingupon the desired results.

FIG. 11 shows a hierarchical architecture for high throughput-low errorsorting according to another embodiment of the present invention. Theillustrated embodiment is a two-stage particle sorting system 800 forsorting a plurality of parallel particles streams in a first stage,aggregating the outputs of the first stage and then performing asecondary sorting process on the output of the first stage. An inputstream of particles in suspension 80 from a particle input chamber 88 issplit among N single sorting channels 81 a-81 n, each channel beingcapable of sorting a selected number of particles per second. Eachchannel 81 includes a detection region 84 for examining the particlesand identifying particles that have a predetermined characteristic, anda switching region 82 for separating the particles having thepredetermined characteristic from the other particles in the stream, asdescribed above. The switching region 82 produces two output streams ofparticles: a “selected” stream and a “rejected” stream in its switchingregion 82 based on the measured particle characteristics at thedetection region 84. The “selected” streams from each channel areaggregated in an aggregation region 86 into one stream to be sortedagain in a secondary sorting channel 810. As shown, the secondarysorting channel 810 repeats the sorting process of detecting and sortingbased on a predetermined characteristic.

Given that each single channel sorting process produces some error (y)rate (y is a probability less than one of a particle being “selected” bymistake) of mistaken selections, the hierarchical architecture producesan lower error rate of y² for a 2-stage hierarchy as drawn or y^(n) foran n-stage hierarchy. For example, if the single channel error rate is1% the 2-stage error rate is 0.01% or one part in 10⁴.

Alternatively, the architecture could have M primary sets of N sortingchannels per secondary channel. Given that the application wants tocapture particles that have a presence in the input at rate z and singlechannel sorters have a maximum sorting rate x particles per second. Thesystem throughput is M*N*x in particles per second. The number ofparticles aggregated in N channels per second is N*x*z and so N*z mustbe less than 1 so that all particles aggregated from N channels can besorted by a single secondary channel. To increase throughput above N=1/zone must add parallel groups of N primary +1 secondary channels. Overallthroughput then comes from M*N*x with M secondary channels.

FIG. 12 show a parallel-serial particle sorting system 160 according toanother embodiment of the invention. The parallel-serial particlesorting system 160 includes a first parallel sorting module 161 and asecond parallel sorting module 162. The first sorting module 161 isapplied in multiple marked particles and particles having both markersare sorted out and conveyed through the exit channel 165.

FIG. 13 shows another parallel-serial particle sorting system 170. Thefirst parallel sorting module 171 separates particles having a firstmarker, collects the particles from the different channels and conveysthe particles having the first marker through the first exit channel175. All other particles are then fed into a second parallel sorter 172for sorting particles having a second marker. The particles having thesecond marker are collected and conveyed through a second exit channel176. Particles having neither the first marker nor the second marker areconveyed through a third exit channel 177.

According to one embodiment of the invention, shown in FIGS. 14 a and 14b, the particle sorting system may include sensors for measuringvelocity, location and/or size of particles. The measurement ofvelocity, location and/or size may be made simultaneously withclassification of the particles for sorting or at a different time. Inparallel channel based systems, as shown in FIG. 11, the differentchannels may have different flow resistances, causing the velocity ofthe particles or cells in each channel to be different. In systems wherethe detection region 84 is separated from the switching region 82 by adistance L, the velocity of the particles in the channel 81 must beknown in order to set the switching time delay T (i.e., the time todelay switch actuation relative to the moment of detection of a targetparticle).

In most optical systems for detecting cells or particles, the region inwhich the cell creates light on the photo detector in the detectionregion will have a much greater size than the size of a cell diameter.Therefore, when light is detected in the detection region, the cell maybe anywhere in the region, making it difficult to pinpoint the exactlocation of the cell. To provide more accurate detection, many pixels ofan optical detector could be packed across the detection region, butthis would have a large cost and require complex support electronics.

According to an illustrative embodiment of the invention, an opticalmask 140 may be added to the detection region to provide accuratevelocity detection by depositing a “masking pattern” directly on thesorting chip. The masking patterns can be deposited so that an edge inthe masking pattern is precisely located (to <1 um precision withcurrent technology) relative to the cell sorting actuator region 82. Asingle optical detector catching light from the cell in the detectionregion 84 will see light when the cell is not masked. The duration ofthe light being turned off by one of the connected opaque parts “bars”of the mask of known length gives a measurement of velocity.

A mask pattern that has several bars 141 of size ranging from 10 um to30 um in 1 um steps results in only bars of size larger than the cellminimizing the signal from the cell. Therefore, such a pattern can alsobe used to measure the size of the cell independently of its signal.Such a ‘gradient mask” also produces a pattern in the optical detectorthat can be analyzed to measure velocity several times for reducing thevariance in the velocity estimate. The pattern in the light induced bythe mask 140 also allows the detector to identify each edge in the mask140. If the bars 141 were all the same, the light signal for each barwould be the same, and one could only tell them apart by sequence.Therefore, a gradient mask pattern will allow a single detector lookingat a broad region (several times the size of a cell) to measure thevelocity of the cell, measure the exact position inside the detectionregion 84 with about 1 um precision relative to the channel structuresand the actuator location on chip and identify the size of the cell toprecision given by the gradient pattern. The gradient mask 140 allowsthe detector to measure these parameters independent of themagnification of the optical system or the nature of the opticaldetector itself.

One skilled in the art will recognize that other devices for measuringthe size, position and or velocity of a particle in the sorting systemin accordance with the teachings of the invention. Suitable devices arereadily available and known to those of ordinary skill in the art.

According to another embodiment, shown in FIG. 15, the particle sortingsystem comprises an array 8000 of non-identical sorting channels. Theuse of a parallel array comprising a series of non-identical sorterchannels 810 a-810 n is more efficient in terms of space, use of opticalpower and adaptation to optimal external actuators. Since the velocityof particles can be accurately sensed using a sensor as described above,the channels do not require a fixed delay between the detection of aproperty and actuation of a switch to deflect a particle having thedetected property. Therefore, certain parameters of the channel, such asthe distance L between a detector 84 and a switch 82 or the shape of thepath between the detector 84 and the switch 82 can be varied.

Using a single laser for each wavelength optical illumination directedperpendicular to the chip, the laser is required to illuminate an areadefined by: (number of channels)×((channel width at detectionregion)+(inter channel spacing C)) (See FIG. 15). However, the activearea where light can be absorbed to create fluorescence is only the areaof the channels: (number of channels)×(channel width), which leaves afill factor of: (channel width)/(channel width+C). The fill factor ispreferably close to 100% to avoid wasting available input light.

Therefore, minimizing the interchannel spacing in a parallel sortingsystem is important to the optical detection region and optical systemefficiency. In the variable array design of the present invention, shownin FIG. 16, the spacing of the channels in the detection region 84approaches the width of the channels, so that light utilizationapproaches about 50%. The channel spacing in the actuation region 82 maybe larger, as shown in FIG. 16. The location of actuators 26 along thechannel may also be varied to make a larger available radius forexternal driver actuators.

The variable array 8000 may also include meanders in selected channelsfor balancing flow resistances of all the channels so that given aconstant pressure drop across all the channels the velocities ofparticles are nearly matched. These can be added either upstream ordownstream of the illustrated system, i.e., on in the region between thedetectors and actuators. As the lengths Li between each channel'sdetection region 821 and its actuator 26 i is known from the design, themeasurement of the particle velocity at the same time as thedetermination regarding which particles to keep provides an improvedcell sorting system.

FIG. 17 illustrates a particle sorting system 1700 according to yetanother embodiment of the invention. The particle sorting system 1700includes a plurality of sorting modules 1701 operating in parallel. Thesystem 1700 includes an input region 1710 for introducing samples toeach sorting module and a detection region 1720 for measuring apredetermined characteristic of particles each sorting channel 1702 inthe detection region. The system also includes a switch region 1730,including an actuator in each sorting module for separating particleshaving a predetermined characteristic from particles that do not havethe predetermined characteristic. As shown, in the embodiment of FIG.17, the sorting channels 1702 distance between each sorting channel inthe detection region 1720 is less than the inter-channel distance in theswitch region 1730. The close spacing in the detection region enablescost saving when a laser is used to detect the particles, while the moredistant separation in the switch region 1730 accommodates various sizedactuators.

The particle sorting system 1700 may also include a secondary sortingmodule 1740 for repeating the sorting process of detecting and sortingbased on a predetermined characteristic to increase the accuracy of thesorting process. According to one embodiment, the system may include anenrichment region 1750 between the array of primary sorting modules 1701and the secondary sorting module 1740 for transitioning the particlesfrom the primary sorting process to the secondary sorting process.According to an illustrative embodiment, the enrichment region 1750transitions the particles by removing excess carrier fluid from theparticles before passing the particles to the secondary sorting module1740. The enrichment region 1750 may also include a hydration device foradding secondary sheet fluid to the particles after enrichment. Theenrichment region 1750 may comprise a membrane inserted into outletchannel 1703, an enrichment channel intersecting the outlet channel 1703and a membrane separating the outlet channel from the enrichmentchannel. Excess carrier fluid is removed from the stream of selectedparticles in the outlet channel 1703 through the membrane and into theenrichment channel before passing the selected particles into thesecondary sorting module 1740.

A suitable system for forming the enrichment region is described inAttorney Docket No. TGZ-023, filed on even date herewith, the contentsof which are herein incorporated by reference.

According to the illustrative embodiment, the removed carrier fluid maybe recycled and fed back into the inlet of the primary channels. Arecycling channel or other device may connect the enrichment region tothe primary channel to allow re-use of the carrier fluid for subsequentsorting process. Alternatively, the carrier fluid may be removed fromrejected particles and introduced into the primary channel inlets priorto discarding the rejected particles.

The present invention has been described relative to an illustrativeembodiment. Since certain changes may be made in the above constructionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description or shown in theaccompanying drawings be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are to cover allgeneric and specific features of the invention described herein, and allstatements of the scope of the invention which, as a matter of language,might be said to fall therebetween.

1-10. (canceled)
 11. A particle processing system comprising: amicrofluidic network including one or more microchannels formed in asubstrate; a first particle processing mechanism associated with a firstflow path of the microfluidic network and adapted for processing oranalyzing a sample having one or more particles suspended in asuspension medium flowing through the first flow path of themicrofluidic network; and a recirculation channel formed in thesubstrate and configured to receive an output from the first processingmechanism including a portion or component of the analyzed or processedsample and introduce the portion or component of the analyzed orprocessed sample as an input to the microfluidic network, wherein therecirculation channel fluidically couples the output from the firstparticle processing mechanism to the input to the microfluidic network.12. The particle processing system of claim 11, wherein the input to themicrofluidic network is a recirculation input to the first particleprocessing mechanism.
 13. The particle processing system of claim 11:wherein the input to the microfluidic network is a recirculation inputto a second particle processing mechanism associated with a second flowpath of the microfluidic network; wherein the second flow path isparallel to the first flow path; and whereby the first and secondparticle processing mechanisms are configured for parallel processing.14. The particle processing system of claim 11, wherein the portion orcomponent of the analyzed or processed sample is a portion or componentof the particle suspension medium.
 15. The particle processing system ofclaim 11 further comprising, an isolating mechanism operatively coupledrelative to the output from the particle processing mechanism adaptedfor isolating the portion or component of the analyzed or processedsample from the output.
 16. The particle processing system of claim 11,wherein the microfluidic network comprises a plurality of microfluidicnetworks.
 17. A particle processing method comprising: flowing a samplehaving one or more particles suspended in a suspension medium through afirst flow path of a microfluidic network, the microfluidic networkincluding one or more microchannels formed in a substrate; processing oranalyzing the sample using a first processing mechanism associated withthe first flow path of the microfluidic network; receiving an outputfrom the first processing mechanism including a portion or component ofthe analyzed or processed sample; and introducing the portion orcomponent of the analyzed or processed sample as an input to themicrofluidic network, wherein the output from the first particleprocessing mechanism is fluidically coupled to the input to themicrofluidic network via a recirculation channel formed in thesubstrate.
 18. The particle processing method of claim 17, wherein theinput to the microfluidic network is a recirculation input to the firstparticle processing mechanism.
 19. The particle processing method ofclaim 17: wherein the input to the microfluidic network is arecirculation input to a second particle processing mechanism associatedwith a second flow path of the microfluidic network; wherein the secondflow path is parallel to the first flow path; and whereby the first andsecond particle processing mechanisms are configured for parallelprocessing.
 20. The particle processing method of claim 17, wherein theportion or component of the analyzed or processed sample is a portion orcomponent of the particle suspension medium.
 21. The particle processingmethod of claim 17 further comprising, isolating the portion orcomponent of the analyzed or processed sample from the output.
 22. Theparticle processing method of claim 17, wherein the microfluidic networkcomprises a plurality of microfluidic networks.
 23. A particleprocessing method comprising: flowing a sample having one or moreparticles suspended in a suspension medium through a first flow path ofa microfluidic network associated with a particle processing system, themicrofluidic network including one or more microchannels formed in asubstrate; performing a process or analysis on the sample using a firstprocessing mechanism associated with the first flow path of themicrofluidic network; and repeating the same process or analysis on aportion or component of an output from the first processing mechanismusing the same particle processing system.
 24. The particle processingmethod of claim 23, wherein a second particle processing mechanismdifferent than the first particle processing mechanism is used to repeatthe same process or analysis on the portion or component of the outputfrom the first processing mechanism.
 25. The particle processing methodof claim 23, wherein the process or analysis is a sorting process basedon a set of criteria and wherein the same process or analysis is thesame sorting process based on the same set of criteria.
 26. The particleprocessing method of claim 25, wherein the repeating the same sortprocess achieves higher purity in a sorted sample.
 27. The particleprocessing method of claim 25, wherein the repeating the same sortprocess achieves higher yield in a sorted sample.
 28. The particleprocessing method of claim 25, wherein the repeating the same process oranalysis enables higher throughput of the sample.
 29. The particleprocessing method of claim 23, wherein the process or analysis isoptically based.
 30. The particle processing method of claim 23, whereinthe process or analysis is magnetically based.